Staphylococcus aureus is an important pathogen associated with various infections, including foodborne outbreaks worldwide. Due to possible contamination during food preparation by food handlers, the presence of this species in kitchens environmental is a risk to global health, mainly S. aureus biofilm-producing. Moreover, there are few reports of the evaluation of biofilm production of S. aureus obtained from food handlers in Brazil. The study aimed to characterize phenotypically and genotypically biofilm production associated with ica-dependent and ica-independent genes in S. aureus isolated from food handlers in this country. A total of 74 Staphylococcus sp. strains were isolated and 37 (50%) were identified as S. aureus through the sau gene detection. All S. aureus strains exhibited a high ability to form biofilm and detected several genes involved in this process, spotlighting the icaA (86.5%), icaD (86.5%), sasG (78.7%), atlA (78.7%) and aap (78.7%). Furthermore, the predominance of biofilm-associated genes in the strong biofilm-producing isolates by TB and TCP was found, presenting a significant association between methods and genes detected (p = < 0,05). In conclusion, the food handlers are essential to food security, which can be reservoirs of important opportunistic pathogens, including S. aureus biofilm-producing, triggering the transmission of this genus isolated worldwide.